Hypertriglyceridemia is a major risk factor for cardiovascular disease (CVD), and is associated with insulin resistance. Rapamycin (RAPA) is a potent antiproliferative and immunosuppressive drug that reduces acute graft rejection. RAPA, however, induces hyperlipidemia by unknown mechanisms. Understanding how the Mammalian Target of Rapamycin (mTOR) pathway regulates insulin signaling and triggers hyperlipidemia will help reduce risk of CVD, and insulin resistance. The hypothesis tested is that RAPA induces hypertriglyceridemia via an insulin-dependent signaling pathway. Rapamycin interferes with insulin-mediated triglycerides storage in adipocytes leading to increased release of free fatty acids (FFA) to the circulation. Subsequently, influx of FFA lead to augmented hepatic secretion of VLDL-triglycerides and hypertriglyceridemia. Supportive evidence is provided by our preliminary studies in renal transplant patients, as well as observations that RAPA abolishes insulin signals via the FK506 binding protein (FKBP)/mammalian target of rapamycin (mTOR)/p70s6kinase pathway (13-17).SPECIFIC AIM 1. To characterize in vitro the molecular component(s) of the signal transduction of rapamycin in insulin-responsive 3T3-L1 adipocytes. Studies will utilize both genome-based and biochemical approaches to define the cellular pathways of RAPA-induced signals in 3T3-L1 adipocytes. Gene expression will be analyzed by pathway-specific cDNA microarray and will be correlated to protein levels and phosphorylation state; phosphatase (PP2A) activities; FFA; TG levels; hormone sensitive lipase (HSL), and lipoprotein lipase (LPL) activities.SPECIFIC AIM 2. To determine in vitro if inhibition of mTOR expression will eliminate RAPA-induced effects via an insulin-dependent pathway. Studies will investigate the functional significance of blocking mTOR translation by antisense oligonucleotides in 3T3-L1 adipocytes treated with RAPA, compared to the controls, on the same parameters determined in aim 1. SPECIFIC AIM 3. To define in vivo the mechanisms of RAPA-induced hypertriglyceridemia, and impact of RAPA on hepatic triglyceride secretion, lipid and lipoprotein regulatory enzymes in guinea pigs. Animals will be injected with either RAPA or vehicle and will be used in two parallel studies to determine the effect of RAPA on hepatic triglyceride secretion, insulin signaling and lipid metabolism. Findings from these studies will identify the intermediate molecules altered by rapamycin and will address the cross-talk between mTOR and insulin signaling.